The study of the recognition or lack of recognition of cancer cells by a host organism has proceeded in many different directions. Understanding of the field presumes some understanding of both basic immunology and oncology.
Early research on mouse tumors revealed that these displayed molecules which led to rejection of tumor cells when transplanted into syngeneic animals. These molecules are “recognized” by T-cells in the recipient animal, and provoke a cytolytic T-cell response with lysis of the transplanted cells. This evidence was first obtained with tumors induced in vitro by chemical carcinogens, such as methylcholanthrene. The antigens expressed by the tumors and which elicited the T-cell response were found to be different for each tumor. See Prehn, et al., J. Natl. Canc. Inst. 18:769-778 (1957); Klein et al., Cancer Res. 20:1561-1572 (1960); Gross, Cancer Res. 3:326-333 (1943), Basombrio, Cancer Res. 30:2458-2462 (1970) for general teachings on inducing tumors with chemical carcinogens and differences in cell surface antigens. This class of antigens has come to be known as “tumor specific transplantation antigens” or “TSTAs.” Following the observation of the presentation of such antigens when induced by chemical carcinogens, similar results were obtained when tumors were induced in vitro via ultraviolet radiation. See Kripke, J. Natl. Canc. Inst. 53:333-1336 (1974).
While T-cell mediated immune responses were observed for the types of tumor described supra, spontaneous tumors were thought to be generally non-immunogenic. These were therefore believed not to present antigens which provoked a response to the tumor in the tumor carrying subject. See Hewitt, et al., Brit. J. Cancer 33:241-259 (1976). The family of tum antigen presenting cell lines are immunogenic variants obtained by mutagenesis of mouse tumor cells or cell lines, as described by Boon et al., J. Exp. Med. 152:1184-1193 (1980), the disclosure of which is incorporated by reference. To elaborate, turn antigens are obtained by mutating tumor cells which do not generate an immune response in syngeneic mice and will form tumors (i.e., “tum+” cells). When these tum+ cells are mutagenized, they are rejected by syngeneic mice, and fail to form tumors (thus “tum−”). See Boon et al., Proc. Natl. Acad. Sci. USA 74:272 (1977), the disclosure of which is incorporated by reference. Many tumor types have been shown to exhibit this phenomenon. See, e.g., Frost et al., Cancer Res. 43:125 (1983). It appears that tum− variants fail to form progressive tumors because they initiate an immune rejection process. The evidence in favor of this hypothesis includes the ability of “tum−” variants of tumors, i.e., those which do not normally form tumors, to do so in mice with immune systems suppressed by sublethal irradiation, Van Pel et al., Proc. Natl. Acad. Sci. USA 76:5282-5285 (1979); and the observation that intraperitoneally injected tum− cells of mastocytoma P815 multiply exponentially for 12-15 days, and then are eliminated in only a few days in the midst of an influx of lymphocytes and macrophages (Uyttenhove et al., J. Exp. Med. 152:1175-1183 (1980)). Further evidence includes the observation that mice acquire an immune memory which permits them to resist subsequent challenge to the same turn variant, even when immunosuppressive amounts of radiation are administered with the following challenge of cells (Boon et al., Proc. Natl. Acad. Sci. USA 74:272-275 (1977); Van Pel et al., supra; Uyttenhove et al., supra). Later research found that when spontaneous tumors were subjected to mutagenesis, immunogenic variants were produced which did generate a response. Indeed, these variants were able to elicit an immune protective response against the original tumor. See Van Pel et al., J. Exp. Med. 157:1992-2001 (1983). Thus, it has been shown that it is possible to elicit presentation of a so-called “tumor rejection antigen” in a tumor which is a target for a syngeneic rejection response. Similar results have been obtained when foreign genes have been transfected into spontaneous tumors. See Fearon et al., Cancer Res. 48:2975-1980 (1988) in this regard.
A class of antigens has been recognized which are presented on the surface of tumor cells and are recognized by cytolytic T cells, leading to lysis. This class of antigens will be referred to as “tumor rejection antigens” or “TRAs” hereafter. TRAs may or may not elicit antibody responses. The extent to which these antigens have been studied, has been via cytolytic T cell characterization studies, in vitro, i.e., the study of the identification of the antigen by a particular cytolytic T cell (“CTL” hereafter) subset. The subset proliferates upon recognition of the presented tumor rejection antigen, and the cells presenting the tumor rejection antigens are lysed. Characterization studies have identified CTL clones which specifically lyse cells expressing the tumor rejection antigens. Examples of this work may be found in Levy et al., Adv. Cancer Res. 24:1-59 (1977); Boon et al., J. Exp. Med. 152:1184-1193 (1980); Brunner et al., J. Immunol. 124:1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 124:1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 12:406-412 (1982); Palladino et al., Cancer. Res. 47:5074-5079 (1987). This type of analysis is required for other types of antigens recognized by CTLs, including minor histocompatibility antigens, the male specific H-Y antigens, and the class of antigens referred to as “tum−” antigens, and discussed herein.
A tumor exemplary of the subject matter described supra is known as P815. See DePlaen et al., Proc. Natl. Acad. Sci. USA 85:2274-2278 (1988); Szikora et al., EMBO J. 9:1041-1050 (1990), and Sibille et al., J. Exp. Med. 172:35-45 (1990), the disclosures of which are incorporated by reference. The P815 tumor is a mastocytoma, induced in a DBA/2 mouse with methylcholanthrene and cultured as both an in vitro tumor and a cell line. The P815 line has generated many tum variants following mutagenesis, including variants referred to as P91A (DePlaen, supra), 35B (Szikora, supra), and P198 (Sibille, supra). In contrast to tumor rejection antigens—and this is a key distinction—the tum antigens are only present after the tumor cells are mutagenized. Tumor rejection antigens are present on cells of a given tumor without mutagenesis. Hence, with reference to the literature, a cell line can be tum+, such as the line referred to as “P11,” and can be provoked to produce turn variants. Since the tum− phenotype differs from that of the parent cell line, one expects a difference in the DNA of tum cell lines as compared to their tum+ parental lines, and this difference can be exploited to locate the gene of interest in tune cells. As a result, it was found that genes of tum variants such as P91A, 35B and P198 differ from their normal alleles by point mutations in the coding regions of the gene. See Szikora and Sibille, supra, and Lurquin et al., Cell 58:293-303 (1989). This has proved not to be the case with the TRAs of this invention. These papers also demonstrated that peptides derived from the tum− antigen are presented by H-2d Class I molecules for recognition by CTLs. P91A is presented by Ld, P35 by Dd and P198 by Kd.
PCT application PCT/US92/04354, filed on May 22, 1992 assigned to the same assignee as the subject application, teaches a family of human tumor rejection antigen precursor coding genes, referred to as the MAGE family. Several of these genes are also discussed in van der Bruggen et al., Science 254:1643 (1991). It is now clear that the various genes of the MAGE family are expressed in tumor cells, and can serve as markers for the diagnosis of such tumors, as well as for other purposes discussed therein. See also Traversari et al., Immunogenetics 35:145 (1992); van der Bruggen et al., Science 254:1643 (1991) and DePlaen, et al., Immunogenetics 40:360 (1994). The mechanism by which a protein is processed and presented on a cell surface has now been fairly well documented. A cursory review of the development of the field may be found in Barinaga, “Getting Some ‘Backbone’:How MHC Binds Peptides,” Science 257:880 (1992); also, see Fremont et al., Science 257:919 (1992); Matsumura et al., Science 257:927 (1992); Engelhard, Ann. Rev. Immunol. 12:181-207 (1994); Madden, et al., Cell 75:693-708 (1993); Ramensee, et al., Ann. Rev. Immunol. 11:213-244(1993); Germain, Cell 76:287-299(1994). These papers generally point to a requirement that the peptide which binds to an MHC/HLA molecule be nine amino acids long (a “nonapeptide”), and to the importance of the second and ninth residues of the nonapeptide. For H-2kb, the anchor residues are positions 5 and 8 of an octamer, for H-2Db, they are positions 5 and 9 of a nonapeptide while the anchor residues for HLA-A1 are positions 3 and 9 of a nonamer. Generally, for HLA molecules, positions 2 and 9 are anchors.
Studies on the MAGE family of genes have now revealed that a particular nonapeptide is in fact presented on the surface of some tumor cells, and that the presentation of the nonapeptide requires that the presenting molecule be HLA-A1. Complexes of the MAGE-1 tumor rejection antigen (the “TRA” or “nonapeptide”) leads to lysis of the cell presenting it by cytolytic T cells (“CTLs”).
Research presented in, e.g., U.S. Pat. No. 5,405,940 filed Aug. 31, 1992, and in U.S. Pat. No. 5,571,711, found that when comparing homologous regions of various MAGE genes to the region of the MAGE-1 gene coding for the relevant nonapeptide, there is a great deal of homology. Indeed, these observations lead to one of the aspects of the invention disclosed and claimed therein, which is a family of nonapeptides all of which have the same N-terminal and C-terminal amino acids. These nonapeptides were described as being useful for various purposes which includes their use as immunogens, either alone or coupled to carrier peptides. Nonapeptides are of sufficient size to constitute an antigenic epitope, and the antibodies generated thereto were described as being useful for identifying the nonapeptide, either as it exists alone, or as part of a larger polypeptide.
The preceding survey of the relevant literature shows that various peptides, usually eight, nine, or ten amino acids in length, complex with MHC molecules and present targets for recognition by cytolytic T cells. A great deal of study has been carried out on melanoma, and melanoma antigens which are recognized by cytolytic T cells are now divided into three broad categories. The first, which includes many of the antigens discussed, supra, (e.g., MAGE), are expressed in some melanomas, as well as other tumor types, and normal testis and placenta. The antigens are the expression product of normal genes which are usually silent in normal tissues.
A second family of melanoma antigens includes antigens which are derived from mutant forms of normal proteins. Examples of this family are MUM-1 (Coulie, et al., Proc. Natl. Acad. Sci. USA 92:7976-7980 (1955)); CDK4 (Wölfel, et al., Science 269:1281-1284(1955)); Bcatenin (Robbins, et al., J. Exp. Med. 183:1185-1192 (1996)); and HLA-A2 (Brandel, et al., J. Exp. Med. 183:2501-2508(1996)). A third category, also discussed, supra, includes the differentiation antigens which are expressed by both melanoma and melanocytes. Exemplary are tyrosinase, gp100, gp75, and Melan A/Mart-1. See U.S. Pat. No. 5,620,886 incorporated by reference, with respect to Melan-A. See Wölfel, et al., Eur. J. Immunol. 24:759 (1994) and Brichard, et al., Eur. J. Immunol. 26:224 (1996) for tyrosinase; Kang, et al., J. Immunol. 155:1343 (1995); Cox, et al., Science 264:716 (1994); Kawakami, et al., J. Immunol. 154:3961 (1995) for gp 100; Wang, et al., J. Exp. Med. 183:1131 (1996)for gp75.
There are several approaches that are available for identifying HLA restricted peptides. For example, Boon, et al., J. Exp. Med. 183:725-729 (1996), describes how to identify peptides targets of CD8+ T cells with reactivity for autologous melanoma cells. The methodology requires transfer of antigen expression to non-expressing cells, via either cosmids or cDNA vectors. See Van der Bruggen, et al., Science 254:1643-1650 (1991); and Kawakami, et al., Proc. Natl. Acad. Sci. USA 91:6458-6492 (1994), respectively, both of which are incorporated by reference. In each case, the transfecting molecule must encode the relevant antigen. Where necessary, an HLA-Class I restriction element can also be used. See DePlaen, et al., Methods 12:125-142 (1997).
When coding sequences for T cell recognized tumor antigens have been defined, HLA bindings motif analysis, such as that provided by Falk, et al., Nature 357:290-296 (1991) can be very useful in identifying relevant peptides.
Hunt et al., Science 255: 1261-1263 (1992) describe a method for identifying peptides by eluting these from HLA molecules, fractionating them via HPLC, and then employing structural identification techniques. Examples of the use of this methodology can be seen in Cox, et al., Science 264:716-719 (1994); Skipper, et al., J. Exp. Med. 183:527-534 (1996); and Castelli, et al., J. Exp. Med. 181:363-368 (1995). There are technical challenges involved in this approach, and it has not been applied widely.
An approach to identifying peptide targets of known tumor antigens which use viral vectors is known. The technique includes inducing a de vovo specific response by naive T cells (Chaux, et al., J. Immunol. 163:2928-2936 (1999); Butterfield, et al., J. Immunol. 161:5607-5613 (1998)); and in stimulating and expanding in vivo sensitized T cells. See, e.g. Toso, et al Canc. Res. 56:16-20 (1996); Yee, et al., J. Immunol. 157:4079-4086 (1996); Kim, et al., J. Immunother. 20:276-286 (1997); Ferrari, et al., Blood 90:2406-2416 (1997). The T cells are then used to identify naturally processed tumor peptides eliciting a T cell response.
One of the drawbacks of the work described supra is the emphasis on HLA-A alleles, particularly HLA-A2 presentation. Very little is known about MHC/HLA restriction for other MHC/HLA molecules. Of the MHC/HLA molecules which are not an HLA-A subtype, the HLA-B27 molecule has been studied most extensively. See, e.g., Parker, et al, J. Immunol. 152:163 (1994), incorporated by reference. Its frequency would suggest that, in a given molecule that is processed to MHC/HLA ligands and/or epitopes, HLA-B27 binders might be expected. As will be shown, however, this was not the case with the invention described herein.
In contrast to HLA-A2 and HLA-B27, information on HLA-C molecules and their binding peptides is scant. Binding motifs are not well characterized, and few peptides have been tested. The frequency of HLA-C occurrence is much lower than the occurrence of HLA-A and B molecules, and the HLA-C molecules are far from the first choice for investigation in a population pool. One of the unexpected findings of the work described herein was the identification of two HLA-C epitopes, as there was little to suggest these in the literature and, as will be elaborated on herein, from the experimental design.
The molecule referred to as “NY-ESO-1”, as described in, e.g., U.S. Pat. No. 5,804,381, incorporated by reference, is recognized as one of the most immunogenic of tumor antigens. Nearly half of patients with advanced cancer express the antigen (Stockert, et al., J. Ex . Med. 187:1349-1354 (1998)), and the expression is accompanied by both a strong CD4+ and a strong CD8+ T cell response. See Jäger, et al., J. Exp. Med. 191:625-630 (2000); Jäger et al., J. Exp. Med. 167:265-270 (1998); Jäger, et al., Proc. Natl. Acad. Sci. USA 97:4760-4765 (2000); Chen, et al., J. Immunol. [press]. Peptides derived from the molecule which are HLA-A2 epitopes are known (Jäger, et al., J. Exp. Med. 187:265-270 (1998)); and Wang et al., J. Immunol. 161:3598-3600 (1998), describes HLA-A31 binding epitopes.
It has now been found that NY-ESO-1 also presents epitopes which bind to HLA-C molecules, such as HLA-Cw3 and HLA-Cw6. See, e.g., p. 7, line 13 after “. . . HLA-Cw3 and HLA-Cw6.” NY-ESO-1 has a homologous sequence to another tumor rejection antigen called LAGE-1 (Lethe et al. U.S. Pat. No. 5,811,519). It follows from what is known about the MAGE-A1/HLA-A1 and MAGE-A3/HLA-A1 peptides that the equivalent regions of LAGE- 1 encoding the relevant nonapeptides would also present epitopes which bind with HLA-C molecules, such as HLA-Cw3 and HLA-Cw6. These peptides, and the ramifications of their discovery, are a part of the invention. Also a part of the invention is the methodology by which they were identified. All facets of the invention are elaborated in the disclosure which follows.